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The cAMP-response element-binding protein (CREB) is a transcription factor located within the cell nucleus. Following phosphorylation at the Ser133 site by various receptor-activated protein kinases—such as Protein Kinase A (PKA), Calmodulin-dependent Protein Kinase (CaMK), and Mitogen-activated Protein Kinase (MAPK)—as well as other kinases, CREB binds to the cAMP-response element (CRE) located within the promoters of target genes. The CRE serves as the primary binding site for CREB and is responsible for its activation. The CRE acts as a focal point for numerous extracellular and intracellular signaling pathways, including those involving cAMP, calcium, GPCRs, and neurotrophic factors. The cAMP/PKA signaling pathway is critical for a wide range of biological processes and organisms. Within the cAMP/PKA pathway, CREB is activated through phosphorylation by PKA and subsequently binds to the CRE, which typically features the consensus motif 5'-TGACGTCA-3'. Given that the CRE functions as a key regulatory element within the cAMP/PKA signaling pathway, it serves as a valuable target for investigating the effects of various inhibitors.

The CRE-Luc CHO-K1 reporter cell line consists of CHO-K1 cells in which the expression of the Luciferase (Luc) reporter gene is under the transcriptional control of the CRE. Forskolin is a naturally occurring compound that acts as a direct activator of adenylyl cyclase (AC), thereby stimulating the cAMP/CREB signaling cascade; the underlying mechanism is illustrated in the figure below.

Figure 1. Schematic Diagram of the CRE-Luc CHO-K1 Principle

Figure 2. Induction of CRE Activity by Forskolin in CRE-Luc CHO-K1(C32)..

Cell Resuscitation
1)Rapidly thaw the frozen cells in a 37 °C water bath for approximately 60 seconds. Once thawed (which may take slightly less or more than 60 seconds), immediately transfer the cell suspension from the cryovial into a 15 mL centrifuge tube containing 10 mL of pre-warmed CHO-K1 Human CRE-Luc Reporter Cell complete culture medium.
2)Centrifuge cells at 1000 rpm for 5 min to remove medium, then resuspend cells in 5 mL of pre-warmed complete medium.
3)Transfer the cell suspension into a T25 culture flask and incubate at 37 °C with 5% CO₂.
4)After approximately 24–36 hours, replace the medium or passage the cells to remove non-adherent dead cells.

Subculturing procedure
1)When the cell density reaches the appropriate confluency for passaging, wash the cells with PBS, then add 1 mL trypsin to detach the cells. When more than 80% of the cells detach upon gently tapping the culture flask, add complete culture medium to terminate digestion. Gently pipette to obtain a single-cell suspension, transfer to a 15 mL centrifuge tube, and centrifuge at 1000 rpm for 5 minutes.

2)Discard supernatant after centrifugation. Resuspend cells in fresh medium to a single-cell suspension and transfer to a new culture flask for continued growth.

Cell Freezing
After trypsinization and centrifugation of cells from each T75 flask or 10 cm culture dish, discard the supernatant. Add 2 mL of cryopreservation medium (90% FBS + 10% DMSO), gently resuspend thoroughly, and aliquot into two cryovials. Immediately place the cryovials into a controlled-rate freezing container (e.g., Nalgene 5100-0001), fill with isopropanol to the indicated level, and store at −80 °C. After 24 hours, transfer the cryovials to liquid nitrogen for long-term storage.